Cytostatin I

ABSTRACT

A human cytostatin I polypeptide and DNA encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for the treatment of cancers, particularly breast cancer, leukemias, and other matastases.

[0001] This application is a divisional of U.S. application Ser. No.09/361,737, filed Jul. 28, 1999, which is a divisional of U.S.application Ser. No. 09/023,073, filed Feb. 13, 1998 (now U.S. Pat. No.5,977,309), which is a divisional of U.S. application Ser. No.08/470,298, filed Jun. 6, 1995 (now U.S. Pat. No. 5,844,081), which is adivisional of U.S. application Ser. No. 08/409,731, filed Mar. 24, 1995(now U.S. Pat. No. 5,658,758), each of which are hereby incorporated byreference herein.

[0002] This invention relates to newly identified polynucleotides,polypeptides encoded by such polynucleotides, the use of suchpolynucleotides and polypeptides, as well as the production of suchpolynucleotides and polypeptides. More particularly, the polypeptide ofthe present invention is cytostatin I, a polypeptide modulating cellularmetabolism. The invention also relates to inhibiting the action of suchpolypeptides.

BACKGROUND

[0003] The cytostatin I of the present invention has been putativelyidentified as a growth inhibitory protein. This identification has beenmade as a result of amino acid sequence homology to mammary-derivedgrowth inhibitor (MDGI) and direct measurements on cell growth.

[0004] Mammary-derived growth inhibitor (MDGI) is a cell growthinhibitor and differentiation factor firstly purified mammary carcinomacells Ehrlich ascites, and then from cows milk and bovine mammary gland(Grosse et al. 2 references). MDGI inhibits proliferation of mammaryepithelial cell lines in a dose-dependent and reversible manner. Maximalinhibition of cell proliferation by purified MDGI is in the range of 35to 50%. In these cells half-maximal inhibition was obtained with about10⁻¹⁰ M MDGI (1 ng/ml). Inhibition was abolished by simultaneouslyadding epidermal growth factor (EGF), insulin. MDGI also inhibits theproliferation of several other permanent mammary carcinoma cell lines.MDGI has been shown to be immunologically related to a fibroblast growthinhibitor.

[0005] Peptides that locally signal growth cessation and stimulatedifferentiation of the developing epithelium are very important formammary gland development. Recombinant and wild-type forms ofmammary-derived growth inhibitor (MDGI) and heart-fatty acid bindingprotein (FABP), which belong to the FABP family, specifically inhibitgrowth of normal mouse mammary epithelial cells (MEC) and promotemorphological differentiation, stimulates its own expression andpromotes milk protein synthesis. Selective inhibition of endogenous MDGIexpression in MEC by antisense phosphorothioate oligonucleotidessuppresses appearance of alveolar end buds and lowers the beta-caseinlevel in organ cultures. Furthermore, MDGI suppresses the mitogeniceffects of EGF, and EGF antagonizes the activities of MDGI. Finally, theregulatory properties of MDGI can be fully mimicked by an 11-amino acidsequence, represented in the COOH terminus of MDGI and a subfamily ofstructurally related FABPs. MDGI is the first known growth inhibitorwhich promotes mammary gland differentiation. The amount of MDGIincreased dramatically with the onset of lactation after delivery.Recent studies shows that a new posttranslational processing form ofMDGI, MDGI 2, not present in lactation, was found in the bovine glandduring pregnancy (Brandt et al, Biochem Biophy Res Comm Vol 189, p406,Nov. 30, 1992). To date, bovine, rat and mouse MDGI have been identifiedbut no human MDGI or MDGI-like protein.

[0006] There is no sequence homology between MDGI and other known growthinhibitors. Thus, along with interferons, transforming growth factors p,and tumor necrosis factors, MDGI is one of the few naturally occurringgrowth inhibitors for mammary epithelium identified so far. Sequenceanalysis revealed extensive sequence homology of MDGI to a family of lowmolecular mass hydrophobic ligand-binding proteins, among them a fattyacid-binding protein (FABP) from brain and heart, myelin P2, adifferentiation associated protein in adipocytes (p422), gastrotropin,and the cellular retinoic acid-binding protein (CRABP). These proteinsbasically share two properties in common: they bind hydrophobic ligandssuch as long-chain fatty acids, retinoids, and eicosanoids, and they areexpressed in a differentiation-dependent manner in mammary gland, heart,liver, brain, or intestine. All these proteins act intracellularlyexcept MDGI and gastrotropin, which act extracellularly in vitro. TheC-terminus of MDGI residues 126-130 are identical to residues 108-112 ofbovine growth hormone. This stretch of amino acids is part of a sequenceof growth hormone that is essential for its biological activity.Synthetic peptides corresponding to the MDGI-sequence, residue 121-131mimic the effects of MDGI. The functions of these MDGI proteins are notyet well-defined, although a role in fatty acid transport,sequestration, or metabolism has been widely discussed. Interaction withas yet unknown hydrophobic ligands might play a functional role in themechanism of growth inhibition excerted by MDGI. It is proposed thatMDGI may act in an autocrine manner as a growth inhibitor, however, MDGIlack a signal sequence for membrane translocation, most of MDGI has anintracellular localization. With regard to the secretion, an analogymight exit to other growth factors that also lack a signal sequence likeFGF and PG-ECGF. In those cases cell damage as a possible way ofsecretion, or the existence of related factors with a signal sequence asa physiological ligands of the respective surface receptors, have beendiscussed.

[0007] Among other activities, MDGI reportedly may inhibit c-fos, c-mycand c-ras expression. MDGI has differentiation-promoting activity onmouse pluripotent embryonic stem cells and supports the commitment ofundifferentiated ESC for neural differentiation. It is also suggestedthat MDGI may be involved in the regulation of endothelial cellproliferation.

[0008] MDGI inhibits the induction of supersensitivity of neonatal ratheart muscle cells for beta-adrenergic receptors by lipoxygenasemetabolites and various agents. The inhibitory activity of MDGI relatedto the induction of supersensitivity for hydrophilic beta-adrenergicagonists might point to a physiological role for a close relative ofMDGI—the cardiac fatty acid-binding protein (H-FABP). One function ofH-FABP could be to protect, the heart, under pathophysiologicalconditions, from lipoxygenase metabolites causing supersensitivity ofbeta-adrenergic receptors. Thus, H-FABP may be a physiological modulatorof beta-adrenergic responses in the cardiac muscle.

[0009] In accordance with one aspect of the present invention, there isprovided a novel mature polypeptide which is Cytostatin I, as well asbiologically active and diagnostically or therapeutically usefulfragments, analogs and derivatives thereof. The polypeptide of thepresent invention is of human origin.

[0010] In accordance with another aspect of the present invention, thereare provided isolated nucleic acid molecules encoding human cytostatinI, including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs andbiologically active and diagnostically or therapeutically usefulfragments thereof.

[0011] In accordance with yet a further aspect of the present invention,there is provided a process for producing such polypeptide byrecombinant techniques comprising culturing recombinant prokaryoticand/or eukaryotic host cells, containing a human cytostatin I nucleicacid sequence, under conditions promoting expression of said protein andsubsequent recovery of said protein.

[0012] In accordance with yet a further aspect of the present invention,there is provided a process for utilizing such polypeptide, orpolynucleotide encoding such polypeptide for therapeutic purposes, forexample, as a cell growth inhibitor and as to cause differentiationstimulatory activity on various responsive types of tissues and cells.

[0013] In accordance with yet a further aspect of the present invention,there is also provided nucleic acid probes comprising nucleic acidmolecules of sufficient length to specifically hybridize to humancytostatin I sequences.

[0014] In accordance with yet a further aspect of the present invention,there are provided antibodies against such cytostatin I polypeptides.

[0015] In accordance with another aspect of the present invention, thereare provided cytostatin I agonists which mimic Cytostatin I and bind tothe cytostatin I receptors to elicit growth inhibitory responses orwhich stimulate differentiation-promoting activity on progenitory celltypes.

[0016] In accordance with yet another aspect of the present invention,there are provided antagonists to such polypeptides, which may be usedto inhibit the action of such polypeptides, for example, in thetreatment of excessive inhibition of cell or tissue growth orinappropriate differentiation stimulatory activity.

[0017] There is a need for a human MDGI-like protein and the geneencoding it. These and other aspects of the present invention should beapparent to those skilled in the art from the teachings herein.

[0018] The following drawings are illustrative of embodiments of theinvention and are not meant to limit the scope of the invention asencompassed by the claims.

SUMMARY

[0019] Human cytostatin I is described for the first time together withits use as in inhibiting growth and stimulating differentiation of humancells. Translated full length cytostatin I coding sequence has goodhomology with mouse mammary-derived growth inhibitor (MDGI). MDGI wasoriginally identified as the cellular retinoic acid-binding protein(CRABP). Both CRABP and MDGI belong to a family of proteins known tobind hydrophobic ligands, referred to as Fatty acid binding proteins(FABPs). Cytostatin I is 33% identical and 63% similar to mouse MDGI.Cytostatin I is highly expressed in spleen and kidney, moderatelyexpressed in liver and thymus. The selective expression of cytostatin Iwas demonstrated during analysis expression in selected human tissues.The cytostatin I gene was found three times in nine week old early stagelibrary, it was found once each in breast lympho node library, pancreaslibrary and tonsils library. Cytostatin I protein was expressed andpurified from E. coli. Our findings demonstrate that cytostatin I hasgrowth inhibitory activity against breast cancer cells, leukemia cells,fibroblast cells, and endothelial cells.

BRIEF DESCRIPTION OF DRAWINGS

[0020]FIG. 1. Nucleotide and amino acid sequence of human Cytostatin I.

[0021] The nucleotide sequence (SEQ ID NO:1) of the cDNA encoding humancytostatin and amino acid sequence (SEQ ID NO:2) is shown. The cDNAsequence encodes a primary translation product of 107 amino acids ofwhich the first 21 to 38 amino acids likely represent a putative leadersequence or transmembrane domain.

[0022] FIGS. 2A-B. Sequence homology of Cytostatin I with other familymembers.

[0023] Comparison of the amino acid sequence of cytostatin I (HTOBH93(SEQ ID NO:2), top) to other members in the family is shown MDGI (SEQ IDNO:7); CRBPI (SEQ ID NO:8); CRBPII (SEQ ID NO:9); FABP (SEQ ID NO:10);and Myelin P2 (SEQ ID NO:11).

[0024]FIG. 3 Tissue distribution of cytostatin I.

[0025] (3A & 3B) Two μg of polyA RNA from the human tissues indicatedwere separated on a 1% agarose-formaldehyde gel and transferred to anylon membrane. The membrane was probed with ³²P-labeled cytostatin IcDNA probe. Cytostatin I is highly expressed in spleen and kidney,moderately expressed in liver and thymus. The lanes on the 3A and 3Bgels are: FIG. 3B Lane 1, spleen heart Lane 2, thymus brain Lane 3,prostate placenta Lane 4, testis lung Lane 5, ovary liver Lane 6, smallintestinal skeletal muscle Lane 7, colon kidney Lane 8, peripheral bloodleukocytes pancreas

[0026] 3C.) 10 μg of total RNA from the cell lines shown were separatedon a 1% agarose-formaldehyde gel and transferred to a nylon membrane.The membrane was probed with ³²P-labeled cytostatin I cDNA. Lane 1,CAMA1 (breast cancer); Lane 2 AN3CA (uterine cancer); Lane 3, SK.UT.1(uterine cancer); Lane 4, MG63 (osteoblastoma); Lane 5, HOS(osteoblastoma); Lane 6, MCF7 (breast cancer); Lane 7, OVCAR-3 (ovariancancer); Lane 8, CAOV-3 (ovarian cancer); Lane 9, HUVEC; Lane 10, AOSMIC(smooth muscle); Lane 11, Fore skin fibroblast. The expression ofcystatin I is undetectable in these cells.

[0027]FIG. 4 Purification of bacterial-expressed human cytostatin I(HG07400-2E).

[0028] The entire coding sequence including the putative signal sequenceor transmembrane domain was fused in frame with a 6-His tag present inthe expression vector pQE9 (Qiagen). E. coli harboring the expressionplasmid were induced with 1 mM IPTG during the logarithmic growth phase.Following a 3-hour induction, the cell pellet was lysed with 6MGuanidine hydrochloride and cytostatin I was purified using aNickel-chelate affinity chromatography column. The highly purifiedprotein was denatured by dialysis in PBS buffer. M, molecular weightmarkers; Lane 1 and 2, induced cell lysate; Lane 3 and 4, uninduced celllysate; Lane 5, pass through fraction from Nickel-chelate columnpurification; Lane 6, 7 and 8, Fraction eluted with 6M Guanidinehydrochloride (pH 5); 9 Fraction eluted with 6M Guanidine hydrochloride(pH 2).

[0029] FIGS. 5A-E

[0030]5A Growth inhibitory activity of cytostatin I (HG07400-1E, highestconcentration 100 ng/ml) against Mdamb 231 human breast cancer cells.

[0031]5B Growth inhibitory activity of cytostatin I (HG07400-2E, highestconcentration 1000 ng/ml) against Mdamb 231 human breast cancer cells.

[0032]5C Growth inhibitory activity of cytostatin I (HG07400-1E) againstJurat human T cell leukemia cells.

[0033]5D Growth inhibitory activity of cytostatin I (HG07400-2E) againstCCD-29LU human lung fibroblast cells.

[0034]5E Growth inhibitory activity of cytostatin I (HG07400-2E) againstCPA 47 bovine pulmonary artery endothelial cells.

[0035] In accordance with an aspect of the present invention, there isprovided an isolated nucleic acid (polynucleotide) which encodes for themature polypeptide having the deduced amino acid sequence of FIG. 1 orfor the mature cytostaatin I polypeptide encoded by the cDNA of theclone deposited with the American Type Culture Collection as ATCC®Deposit No. 97103 on Mar. 21, 1995. The deposit is maintained under theterms of the Budapest Treaty and will be made available to a patentoffice signatory to the Budapest Treaty.

[0036] A polynucleotide encoding a cytostatin I of the present inventionmay be obtained from various human tissues, particularly nine weekembryonic tissue, breast lymph node, pancrease, spleen, kidney, liver,thymus and tonsils. The polynucleotide of this invention was discoveredin a cDNA library derived from human tonsils. It is structurally relatedto: 1) the mammary-derived growth inhibitor (MDGI) family; 2) theheart-fatty acid binding protein (FABP) family; 3) myelin P2differentiation protein; 4) gastropropin; and 5) the the cellularretinoic acid-binding protein (CRABP). It contains an open reading frameencoding a protein of about 107 amino acid residues. The proteinexhibits the highest degree of homology to non human MDGI with 33%identity and 63 % similarity to mouse MDGI. It is also important thatcytostatin I is highly expressed in spleen and kidney, and moderatelyexpressed in liver and thymus. There are 18 highly concerved amino acidsin cytostatin I when compared to other polypeptides with amino sequencesimilarity. The most conserved sequence is the sequence between aminoacids 54 and 60 where 5 of 7 amino acids are highly conserved.

[0037] The polynucleotide of the present invention may be in the form ofRNA or in the form of DNA, which DNA includes cDNA, genomic DNA, andsynthetic DNA. The DNA may be double-stranded or single-stranded, and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. The coding sequence which encodes the mature polypeptide may beidentical to the coding sequence shown in FIG. 1 or that of thedeposited clone or may be a different coding sequence which codingsequence, as a result of the redundancy or degeneracy of the geneticcode, encodes the same mature polypeptide as the DNA of FIG. 1 or thedeposited cDNA.

[0038] The polynucleotide which encodes for the mature polypeptide ofFIG. 1 or for the mature polypeptide encoded by the deposited cDNA mayinclude, but is not limited to: only the coding sequence for the maturepolypeptide; the coding sequence for the mature polypeptide andadditional coding sequence such as a leader or secretory sequence or aproprotein sequence; the coding sequence for the mature polypeptide (andoptionally additional coding sequence) and non-coding sequence, such asintrons or non-coding sequence 5′ and/or 3′ of the coding sequence forthe mature polypeptide.

[0039] Thus, the term “polynucleotide encoding a polypeptide”encompasses a polynucleotide which includes only coding sequence for thepolypeptide as well as a polynucleotide which includes additional codingand/or non-coding sequence.

[0040] The present invention further relates to variants of thehereinabove described polynucleotides which encode for fragments,analogs and derivatives of the polypeptide having the deduced amino acidsequence of FIG. 1 or the polypeptide encoded by the cDNA of thedeposited clone. The variant of the polynucleotide may be a naturallyoccurring allelic variant of the polynucleotide or a non-naturallyoccurring variant of the polynucleotide.

[0041] Thus, the present invention includes polynucleotides encoding thesame mature polypeptide as shown in FIG. 1 or the same maturepolypeptide encoded by the cDNA of the deposited clone as well asvariants of such polynucleotides which variants encode for a fragment,derivative or analog of the polypeptide of FIG. 1 or the polypeptideencoded by the cDNA of the deposited clone. Such nucleotide variantsinclude deletion variants, substitution variants and addition orinsertion variants.

[0042] As hereinabove indicated, the cytostatin I polynucleotide mayhave a coding sequence which is a naturally occurring allelic variant ofthe coding sequence shown in FIG. 1 or of the coding sequence of thedeposited clone. As known in the art, an allelic variant is an alternateform of a polynucleotide sequence which may have a substitution,deletion or addition of one or more nucleotides, which does notsubstantially alter the function of the encoded polypeptide.

[0043] The present invention also includes polynucleotides, wherein thecoding sequence for the mature cytostatin I polypeptide may be fused inthe same reading frame to a polynucleotide sequence which aids inexpression and secretion of a polypeptide from a host cell, for example,a leader sequence which functions as a secretory sequence forcontrolling transport of a polypeptide from the cell. The polypeptidehaving a leader sequence is a preprotein and may have the leadersequence cleaved by the host cell to form the mature form of thepolypeptide. Thus, for example, the polynucleotide of the presentinvention may encode for a mature protein, or for a protein having aprosequence or for a protein having both a prosequence and a presequence(leader sequence).

[0044] The polynucleotides of the present invention may also have thecoding sequence fused in frame to a marker sequence which allows forpurification of the polypeptide of the present invention. The markersequence may be a hexa-histidine tag supplied by a pQE-9 vector toprovide for purification of the mature polypeptide fused to the markerin the case of a bacterial host, or, for example, the marker sequencemay be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells,is used. The HA tag corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

[0045] The present invention further relates to polynucleotides whichhybridize to the hereinabove-described sequences if there is at least50% and preferably 70% identity between the sequences. The presentinvention particularly relates to polynucleotides which hybridize understringent conditions to the hereinabove-described polynucleotides. Asherein used, the term “stringent conditions” means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences. The polynucleotides which hybridize to thehereinabove described polynucleotides in a preferred embodiment encodepolypeptides which retain substantially the same biological function oractivity as the mature polypeptide encoded by the cDNA of FIG. 1 or thedeposited cDNA.

[0046] The deposit(s) referred to herein will be maintained under theterms of the Budapest Treaty on the International Recognition of theDeposit of Micro-organisms for purposes of Patent Procedure. Thesedeposits are provided merely as convenience to those of skill in the artand are not an admission that a deposit is required under 35 U.S.C.§112. The sequence of the polynucleotides contained in the depositedmaterials, as well as the amino acid sequence of the polypeptidesencoded thereby, are incorporated herein by reference and arecontrolling in the event of any conflict with any description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

[0047] The present invention further relates to a cytostatin Ipolypeptide which has the deduced amino acid sequence of FIG. 1 or whichhas the amino acid sequence encoded by the deposited cDNA, as well asfragments, analogs and derivatives of such polypeptide.

[0048] The terms “fragment,” “derivative” and “analog” when referring tothe polypeptide of FIG. 1 or that encoded by the deposited cDNA, means apolypeptide which retains essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

[0049] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide,preferably a recombinant polypeptide.

[0050] The fragment, derivative or analog of the polypeptide of FIG. 1or that encoded by the deposited cDNA may be (i) one in which one ormore of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

[0051] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0052] The term “isolated” means that the material is removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

[0053]

[0054] The present invention also relates to vectors which includepolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

[0055] Host cells are genetically engineered (transduced or transformedor transfected) with the vectors of this invention which may be, forexample, a cloning vector or an expression vector. The vector may be,for example, in the form of a plasmid, a viral particle, a phage, etc.The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying the cytostatin I genes. The cultureconditions, such as temperature, pH and the like, are those previouslyused with the host cell selected for expression, and will be apparent tothe ordinarily skilled artisan.

[0056] The polynucleotides of the present invention may be employed forproducing polypeptides by recombinant techniques. Thus, for example, thepolynucleotide may be included in any one of a variety of expressionvectors for expressing a polypeptide. Such vectors include chromosomal,nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40;bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectorsderived from combinations of plasmids and phage DNA, viral DNA such asvaccinia, adenovirus, fowl pox virus, and pseudorabies. However, anyother vector may be used as long as it is replicable and viable in thehost.

[0057] The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is inserted into anappropriate restriction endonuclease site(s) by procedures known in theart. Such procedures and others are deemed to be within the scope ofthose skilled in the art.

[0058]

[0059] The DNA sequence in the expression vector is operatively linkedto an appropriate expression control sequence(s) (promoter) to directmRNA synthesis. As representative examples of such promoters, there maybe mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phagelambda P_(L) promoter and other promoters known to control expression ofgenes in prokaryotic or eukaryotic cells or their viruses. Theexpression vector also contains a ribosome binding site for translationinitiation and a transcription terminator. The vector may also includeappropriate sequences for amplifying expression.

[0060] In addition, the expression vectors preferably contain one ormore selectable marker genes to provide a phenotypic trait for selectionof transformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

[0061] The vector containing the appropriate DNA sequence as hereinabovedescribed, as well as an appropriate promoter or control sequence, maybe employed to transform an appropriate host to permit the host toexpress the protein.

[0062] As representative examples of appropriate hosts, there may bementioned: bacterial cells, such as E. coli, Streptomyces, Salmonellatyphimurium; fungal cells, such as yeast; insect cells such asDrosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowesmelanoma; adenoviruses; plant cells, etc. The selection of anappropriate host is deemed to be within the scope of those skilled inthe art from the teachings herein.

[0063] More particularly, the present invention also includesrecombinant constructs comprising one or more of the sequences asbroadly described above. The constructs comprise a vector, such as aplasmid or viral vector, into which a sequence of the invention has beeninserted, in a forward or reverse orientation. In a preferred aspect ofthis embodiment, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the sequence.Large numbers of suitable vectors and promoters are known to those ofskill in the art, and are commercially available. The following vectorsare provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen),pBS, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a,pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene)pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid orvector may be used as long as they are replicable and viable in thehost.

[0064] Promoter regions can be selected from any desired gene using CAT(chloramphenicol transferase) vectors or other vectors with selectablemarkers. Two appropriate vectors are pKK232-8 and pCM7. Particular namedbacterial promoters include lacd, lacZ, T3, T7, gpt, lambda P_(R), P_(L)and trp. Eukaryotic promoters include CMV immediate early, HSV thymidinekinase, early and late SV40, LTRs from retrovirus, and mousemetallothionein-1. Selection of the appropriate vector and promoter iswell within the level of ordinary skill in the art.

[0065] In a further embodiment, the present invention relates to hostcells containing the above-described constructs. The host cell can be ahigher eukaryotic cell, such as a mammalian cell, or a lower eukaryoticcell, such as a yeast cell, or the host cell can be a prokaryotic cell,such as a bacterial cell. Introduction of the construct into the hostcell can be effected by calcium phosphate transfection, DEAE-Dextranmediated transfection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

[0066] The constructs in host cells can be used in a conventional mannerto produce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0067] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), the disclosure of which is hereby incorporated byreference.

[0068] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes is increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act on a promoter to increase itstranscription. Examples including the SV40 enhancer on the late side ofthe replication origin bp 100 to 270, a cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers.

[0069] Generally, recombinant expression vectors will include origins ofreplication and selectable markers permitting transformation of the hostcell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiaeTRP1 gene, and a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence. Such promoters can bederived from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heatshock proteins, among others. The heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences, and preferably, a leader sequence capable ofdirecting secretion of translated protein into the periplasmic space orextracellular medium. Optionally, the heterologous sequence can encode afusion protein including an N-terminal identification peptide impartingdesired characteristics, e.g., stabilization or simplified purificationof expressed recombinant product.

[0070] Useful expression vectors for bacterial use are constructed byinserting a structural DNA sequence encoding a desired protein togetherwith suitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and to, if desirable, provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus, although others may also be employedas a matter of choice.

[0071] As a representative but nonlimiting example, useful expressionvectors for bacterial use can comprise a selectable marker and bacterialorigin of replication derived from commercially available plasmidscomprising genetic elements of the well known cloning vector pBR322(ATCC® 37017). Such commercial vectors include, for example, pKK223-3(Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec,Madison, Wis., USA). These pBR322 “backbone” sections are combined withan appropriate promoter and the structural sequence to be expressed.

[0072] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, the selected promoter isinduced by appropriate means (e.g., temperature shift or chemicalinduction) and cells are cultured for an additional period.

[0073] Cells are typically harvested by centrifugation, disrupted byphysical or chemical means, and the resulting crude extract retained forfurther purification.

[0074] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, suchmethods are well know to those skilled in the art.

[0075] Various mammalian cell culture systems can also be employed toexpress recombinant protein. Examples of mammalian expression systemsinclude the COS-7 lines of monkey kidney fibroblasts, described byGluzman, Cell, 23:175 (1981), and other cell lines capable of expressinga compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK celllines. Mammalian expression vectors will comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation site, splice donor and acceptorsites, transcriptional termination sequences, and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice,and polyadenylation sites may be used to provide the requirednontranscribed genetic elements.

[0076] The cytostatin I polypeptide can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the mature protein. Finally,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

[0077] The cytostatin I polypeptides of the present invention may be anaturally purified product, or a product of chemical syntheticprocedures, or produced by recombinant techniques from a prokaryotic oreukaryotic host (for example, by bacterial, yeast, higher plant, insectand mammalian cells in culture). Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated or may be non-glycosylated. Polypeptidesof the invention may also include an initial methionine amino acidresidue.

[0078] The medical relevance and practical use of the cytostatin I ofthe present invention are based upon comparisons with other knownproteins and by experimental analysis. The experimental results suggestthat cytostatin I, as a therapeutic protein, may have the followingmedical applications:

[0079] 1. Anti-tumor: the growth inhibitory activity of cytostatin I maybe used as a therapeutic agent to treat various cancers.

[0080] 2. Anti-angiogenesis: cytostatin I inhibiting fibroblast andendothelial cell growth.

[0081] 3. Anti-metastasis: tumor cells must attract new vessels in orderto grow and metastasize efficiently.

[0082] 4. Stimulation of milk production after childbirth: cytostatin Iinhibits mammary epithelial cell growth and modulation mammary glanddifferentiation, promotes formation of alveolar buds, supportsdevelopment of differentiated lobuloalveoli, and stimulates milk proteinsynthesis and fat droplet accumulation.

[0083] 5. Promoting involution of breast (return of an enlarged breastto normal size after parturition, childbirth) : Antisensephosphorothioate oligonucleotides or antibodies to cytostatin I couldselective inhibition of endogenous cytostatin I expression in mammaryepithelial cells and suppresses appearance of alveolar end buds andlowers the beta-casein level.

[0084] 6. Stimulation of dairy cows milk production or recombinantproteins produced by cows.

[0085] 7. Modulation of beta-adrenergic sensitivity of cardiac myocytes.

[0086] The various potential therapeutic catagories and uses of thecytostatin I include but are not limited to all aspects of the followingareas of medical practice: 1. Oncology, 2. Cardiovascular, 3.Immunology, 4. Hematology, 5. Metabolism, 6. Gynecology and Obstetrics,and 7. Endocrinology.

[0087] Fragments of the full length cytostatin I gene may be used as ahybridization probe for a cDNA library to isolate the full lengthcytostatin I gene and to isolate other genes which have a high sequencesimilarity to the cytostatin I gene or similar biological activity.Probes of this type generally have at least 20 bases. Preferably,however, the probes have at least 30 bases and generally do not exceed50 bases, although they may have a greater number of bases. The probemay also be used to identify a cDNA clone corresponding to a full lengthtranscript and a genomic clone or clones that contain the completecytostatin I gene including regulatory and promotor regions, exons, andintrons. An example of a screen comprises isolating the coding region ofthe cytostatin I gene by using the known DNA sequence to synthesize anoligonucleotide probe. Labeled oligonucleotides having a sequencecomplementary to that of the gene of the present invention are used toscreen a library of human cDNA, genomic DNA or mRNA to determine whichmembers of the library the probe hybridizes to.

[0088] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments and diagnostics to human disease.

[0089] This invention provides a method for identification of thereceptor for the human cytostatin I ligand. The gene encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting (Coligan, etal., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the cytostatin I ligand, and a cDNA librarycreated from this RNA is divided into pools and used to transfect COScells or other cells that are not responsive to the cytostatin I ligand.Transfected cells which are grown on glass slides are exposed to labeledcytostatin I ligand. The cytostatin I ligand can be labeled by a varietyof means including iodination or inclusion of a recognition site for asite-specific protein kinase. Following fixation and incubation, theslides are subjected to auto-radiographic analysis. Positive pools areidentified and sub-pools are prepared and re-transfected using aniterative sub-pooling and re-screening process, eventually yielding asingle clone that encodes the putative receptor. As an alternativeapproach for receptor identification, labeled ligand can bephotoaffinity linked with cell membrane or extract preparations thatexpress the receptor molecule. Cross-linked material is resolved by PAGEand exposed to X-ray film. The labeled complex containing theligand-receptor can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing would be used to design a set of degenerateoligonucleotide probes to screen a cDNA library to identify the geneencoding the putative receptor.

[0090] The method for determining whether a ligand can bind to thecytostatin I receptor comprises transfecting a cell population (onepresumed not to contain the receptor) with the appropriate vectorexpressing the cytostatin I receptor, such that the cell will nowexpress the cytostatin I receptor. A suitable response system isobtained by transfection of the DNA into a suitable host containing thedesired second messenger pathways including cAMP, ion channels,phosphoinositide kinase, or calcium response. Such a transfection systemprovides a response system to analyze the activity of various ligandsexposed to the cell. Ligands chosen could be identified through arational approach by taking known ligands that interact with similartypes of receptors or using small molecules, cell supernatants orextracts or natural products.

[0091] This invention provides a method of screening drugs to identifythose which enhance (agonists) or block (antagonists) interaction ofligand to receptor. An agonist is a compound which increases the naturalbiological functions of cytostatin I, while antagonists eliminate suchfunctions. As an example, a mammalian cell or membrane preparationexpressing the cytostatin I receptor would be incubated with labeledligand in the presence of the drug. The ability of the drug to enhanceor block this interaction could then be measured. Alternatively, theresponse of a known second messenger system following interaction ofligand and receptor would be measured and compared in the presence orabsence of the drug. Such second messenger systems include but are notlimited to, cAMP guanylate cyclase, ion channels or phosphoinositidehydrolysis. (The present invention also relates to an assay foridentifying potential antagonist specific to cytostatin I. An example ofsuch an assay combines cytostatin I and a potential antagonist withmembrane-bound cytostatin I receptors or recombinant cytostatin I underappropriate conditions for a competitive inhibition assay. Cytostatin Ican be labeled, such as by radio activity, such that the number ofcytostatin I molecules bound to the receptor can determine theeffectiveness of the potential antagonist.

[0092] Potential antagonists include an antibody, or in some cases, anoligopeptide, which binds to the polypeptide. Alternatively, a potentialantagonist may be a closely related protein which binds to the receptorsites, however, they are inactive forms of the polypeptide and therebyprevent the action of cytostatin I since receptor sites are occupied.

[0093] Another potential antagonist is an antisense construct preparedusing antisense technology. Antisense technology can be used to controlgene expression through triple-helix formation or antisense DNA or RNA,both of which methods are based on binding of a polynucleotide to DNA orRNA. For example, the 5′ coding portion of the polynucleotide sequence,which encodes for the mature polypeptides of the present invention, isused to design an antisense RNA oligonucleotide of from about 10 to 40base pairs in length. A DNA oligonucleotide is designed to becomplementary to a region of the gene involved in transcription (triplehelix—see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al,Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)),thereby preventing transcription and the production of cytostatin I. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into cytostatin I polypeptide(Antisense—Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988)). The oligonucleotides described above can also be delivered tocells such that the antisense RNA or DNA may be expressed in vivo toinhibit production of cytostatin I.

[0094] Potential antagonists include a small molecule which binds to andoccupies the receptor binding site of the cytostatin I polypeptidethereby making the binding site inaccessible to receptor such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small peptides or peptide-like molecules.

[0095] The antagonists may be employed to treat disease conditionscaused by excess cytostatin I production or activity. The antagonistsmay be employed in a composition with a pharmaceutically acceptablecarrier, e.g., as hereinafter described.

[0096] The polypeptides of the present invention may be employed incombination with a suitable pharmaceutical carrier. Such compositionscomprise a therapeutically effective amount of the polypeptide, and apharmaceutically acceptable carrier or excipient. Such a carrierincludes but is not limited to saline, buffered saline, dextrose, water,glycerol, ethanol, and combinations thereof. The formulation should suitthe mode of administration.

[0097] The invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration. Inaddition, the polypeptides of the present invention may be employed inconjunction with other therapeutic compounds.

[0098] The pharmaceutical compositions may be administered in aconvenient manner such as by the oral (when protected from hydrolysis ordigestion), topical, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes. The pharmaceuticalcompositions are administered in an amount which is effective fortreating and/or prophylaxis of the specific indication. In general, theyare administered in an amount of at least about 10 μg/kg body weight andin most cases they will be administered in an amount not in excess ofabout 8 mg/Kg body weight per day. In most cases, the dosage is fromabout 10 μg/kg to about 1 mg/kg body weight daily, taking into accountthe routes of administration, symptoms, etc.

[0099] The cytostatin I polypeptides and agonists and antagonists whichare polypeptides may also be employed in accordance with the presentinvention by expression of such polypeptides in vivo, which is oftenreferred to as “gene therapy.”

[0100] Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with theengineered cells then being provided to a patient to be treated with thepolypeptide. Such methods are well-known in the art and are apparentfrom the teachings herein. For example, cells may be engineered by theuse of a retroviral plasmid vector containing RNA encoding a polypeptideof the present invention.

[0101] Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by, for example, procedures known in the art. Forexample, a packaging cell is transduced with a retroviral plasmid vectorcontaining RNA encoding a polypeptide of the present invention such thatthe packaging cell now produces infectious viral particles containingthe gene of interest. These producer cells may be administered to apatient for engineering cells in vivo and expression of the polypeptidein vivo. These and other methods for administering a polypeptide of thepresent invention by such method should be apparent to those skilled inthe art from the teachings of the present invention.

[0102] Retroviruses from which the retroviral plasmid vectorshereinabove mentioned may be derived include, but are not limited to,Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruses suchas Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus,gibbon ape leukemia virus, human immunodeficiency virus, adenovirus,Myeloproliferative Sarcoma Virus, and mammary tumor virus. In oneembodiment, the retroviral plasmid vector is derived from Moloney MurineLeukemia Virus.

[0103] The vector includes one or more promoters. Suitable promoterswhich may be employed include, but are not limited to, the retroviralLTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoterdescribed in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990(1989), or any other promoter (e.g., cellular promoters such aseukaryotic cellular promoters including, but not limited to, thehistone, pol III, and β-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,thymidine kinase (TK) promoters, and B19 parvovirus promoters. Theselection of a suitable promoter will be apparent to those skilled inthe art from the teachings contained herein.

[0104] The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

[0105] The retroviral plasmid vector is employed to transduce packagingcell lines to form producer cell lines. Examples of packaging cellswhich may be transfected include, but are not limited to, the PE501,PA317, _(—)−2, _−AM, PA12, T19-14X, VT-19-17-H2, _CRE, _CRIP, GP+E-86,GP+envAm12, and DAN cell lines as described in Miller, Human GeneTherapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein byreference in its entirety. The vector may transduce the packaging cellsthrough any means known in the art. Such means include, but are notlimited to, electroporation, the use of liposomes, and CaPO₄precipitation. In one alternative, the retroviral plasmid vector may beencapsulated into a liposome, or coupled to a lipid, and thenadministered to a host.

[0106] The producer cell line generates infectious retroviral vectorparticles which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles then may be employed, totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

[0107] This invention is also related to the use of the cytostatin Igene as a diagnostic. Detection of a mutated form of cytostatin I willallow a diagnosis of a disease or a susceptibility to a disease whichresults from underexpression of cytostatin I for example, failure toproperly inhibit growth of a tumor cell.

[0108] Individuals carrying mutations in the human cytostatin I gene maybe detected at the DNA level by a variety of techniques. Nucleic acidsfor diagnosis may be obtained from a patient's cells, such as fromblood, urine, saliva, tissue biopsy and autopsy material. The genomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324:163-166 (1986)) prior toanalysis. RNA or cDNA may also be used for the same purpose. As anexample, PCR primers complementary to the nucleic acid encodingcytostatin I can be used to identify and analyze cytostatin I mutations.For example, deletions and insertions can be detected by a change insize of the amplified product in comparison to the normal genotype.Point mutations can be identified by hybridizing amplified DNA toradiolabeled cytostatin I RNA or alternatively, radiolabeled cytostatinI antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

[0109] Sequence differences between the reference gene and genes havingmutations may be revealed by the direct DNA sequencing method. Inaddition, cloned DNA segments may be employed as probes to detectspecific DNA segments. The sensitivity of this method is greatlyenhanced when combined with PCR. For example, a sequencing primer isused with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

[0110] Genetic testing based on DNA sequence differences may be achievedby detection of alteration in electrophoretic mobility of DNA fragmentsin gels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al, Science, 230:1242 (1985)).

[0111] Sequence changes at specific locations may also be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401(1985)).

[0112] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

[0113] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations can also be detected by in situ analysis.

[0114] The present invention also relates to a diagnostic assay fordetecting altered levels of cytostatin I protein in various tissuessince an over-expression of the proteins compared to normal controltissue samples can detect the presence of cytostatin I. Assays used todetect levels of cytostatin I protein in a sample derived from a hostare well-known to those of skill in the art and includeradioimmunoassays, competitive-binding assays, Western Blot analysis andpreferably an ELISA assay. An Elisa assay initially comprises preparingan antibody specific to the cytostatin I antigen, preferably amonoclonal antibody. In addition a reporter antibody is prepared againstthe monoclonal antibody. To the reporter antibody is attached adetectable reagent such as radioactivity, fluorescence or in thisexample a horseradish peroxidase enzyme. A sample is now removed from ahost and incubated on a solid support, e.g. a polystyrene dish, thatbinds the proteins in the sample. Any free protein binding sites on thedish are then covered by incubating with a non-specific protein such asbovine serum albumin. Next, the monoclonal antibody is incubated in thedish during which time the monoclonal antibodies attach to anycytostatin I proteins attached to the polystyrene dish. All unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is now placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound tocytostatin I. Unattached reporter antibody is then washed out.Peroxidase substrates are then added to the dish and the amount of colordeveloped in a given time period is a measurement of the amount ofcytostatin I protein present in a given volume of patient sample whencompared against a standard curve.

[0115] A competition assay may be employed wherein antibodies specificto cytostatin I is attached to a solid support and labeled cytostatin Iand a sample derived from the host are passed over the solid support andthe amount of label detected attached to the solid support can becorrelated to a quantity of cytostatin I in the sample.

[0116] The sequences of the present invention are also valuable forchromosome identification. The sequence is specifically targeted to andcan hybridize with a particular location on an individual humanchromosome. Moreover, there is a current need for identifying particularsites on the chromosome. Few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with genes associated with disease.

[0117] Briefly, sequences can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region of the gene is used to rapidly select primers thatdo not span more than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

[0118] PCR mapping of somatic cell hybrids is a rapid procedure forassigning a particular DNA to a particular chromosome. Using the presentinvention with the same oligonucleotide primers, sublocalization can beachieved with panels of fragments from specific chromosomes or pools oflarge genomic clones in an analogous manner. Other mapping strategiesthat can similarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

[0119] Fluorescence in situ hybridization (FISH) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with cDNAas short as 500 or 600 bases; however, clones larger than 2,000 bp havea higher likelihood of binding to a unique chromosomal location withsufficient signal intensity for simple detection. FISH requires use ofthe clones from which the express sequence tag (EST) was derived, andthe longer the better. For example, 2,000 bp is good, 4,000 is better,and more than 4,000 is probably not necessary to get good results areasonable percentage of the time. For a review of this technique, seeVerma et al., Human Chromosomes: a Manual of Basic Techniques, PergamonPress, New York (1988).

[0120] Once a sequence has been mapped to a precise chromosomallocation, the physical position of the sequence on the chromosome can becorrelated with genetic map data. Such data are found, for example, inV. McKusick, Mendelian Inheritance in Man (available on line throughJohns Hopkins University Welch Medical Library). The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion are then identified through linkage analysis (coinheritance ofphysically adjacent genes).

[0121] Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

[0122] With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

[0123] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0124] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0125] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler and Milstein,1975, Nature, 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies(Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, AlanR. Liss, Inc., pp. 77-96).

[0126] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice may be used to express humanized antibodies toimmunogenic polypeptide products of this invention.

[0127] The present invention will be further described with reference tothe following examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

[0128] In order to facilitate understanding of the following examplescertain frequently occurring methods and/or terms will be described.

[0129] “Plasmids” are designated by a lower case p preceded and/orfollowed by capital letters and/or numbers. The starting plasmids hereinare either commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed are known in the art and will be apparent to the ordinarilyskilled artisan.

[0130] “Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

[0131] Size separation of the cleaved fragments is performed using 8percent polyacrylamide gel described by Goeddel, D. et al., NucleicAcids Res., 8:4057 (1980).

[0132] “Oligonucleotides” refers to either a single strandedpolydeoxynucleotide or two complementary polydeoxynucleotide strandswhich may be chemically synthesized. Such synthetic oligonucleotideshave no 5′ phosphate and thus will not ligate to another oligonucleotidewithout adding a phosphate with an ATP in the presence of a kinase. Asynthetic oligonucleotide will ligate to a fragment that has not beendephosphorylated.

[0133] “Ligation” refers to the process of forming phosphodiester bondsbetween two double stranded nucleic acid fragments (Maniatis, T., etal., Id., p. 146). Unless otherwise provided, ligation may beaccomplished using known buffers and conditions with 10 units of T4 DNAligase (“ligase”) per 0.5 μg of approximately equimolar amounts of theDNA fragments to be ligated.

[0134] Unless otherwise stated, transformation was performed asdescribed in the method of Graham, F. and Van der Eb, A., Virology,52:456-457 (1973).

EXAMPLE 1 Bacterial Expression and Purification of Cytostatin I

[0135] The DNA sequence encoding cytostatin I, ATCC® #97103, isinitially amplified using PCR oligonucleotide primers corresponding tothe 5′ sequences of the processed cytostatin I protein (minus the signalpeptide sequence) and the vector sequences 3′ to the cytostatin I gene.Additional nucleotides corresponding to the primers used were added tothe 5′ and 3′ sequences respectively. The 5′ oligonucleotide primer hasthe sequence 5′CGCGGATCCATGCCTCCCAACCTCACTG 3′ (SEQ ID NO:3) andcontains a BamHI restriction enzyme site followed by 19 nucleotides ofcytostatin I coding sequence starting from the starting codon of thegene. The 3′ sequence 5′GCGTCTAGACTATCTGACCTTCCTGAAGAC3′ (SEQ ID NO:4)contains complementary sequences to XbaI site and is followed by 20nucleotides of cytostatin I including a stop codon. The restrictionenzyme sites correspond to the restriction enzyme sites on the bacterialexpression vector pQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth,Calif., 91311). pQE-9 encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-9 was then digested with BamHI and XbaI.The amplified sequences were ligated into pQE-9 and were inserted inframe with the sequence encoding for the histidine tag and the RBS. Theligation mixture was then used to transform E. coli strain M15 availablefrom Qiagen under the trademark M15/rep 4 by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacd repressor and also conferskanamycin resistance (Kan^(r)). Transformants are identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis. Clones containing the desired constructs were grown overnight(O/N) in liquid culture in LB media supplemented with both Amp (100ug/ml) and Kan (25 ug/ml). The O/N culture is used to inoculate a largeculture at a ratio of 1:100 to 1:250. The cells were grown to an opticaldensity 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(“Isopropyl-B-D-thiogalacto pyranoside”) was then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacd repressor,clearing the P/O leading to increased gene expression. Cells were grownan extra 3 to 4 hours. Cells were then harvested by centrifugation. Thecell pellet was solubilized in the chaotropic agent 6 Molar GuanidineHCl. After clarification, solubilized cytostatin I was purified fromthis solution by chromatography on a Nickel-Chelate column underconditions that allow for tight binding by proteins containing the 6-Histag (Hochuli, E. et al., J. Chromatography 411:177-184 (1984)).Cytostatin I (90% pure) was eluted from the column in 6 molar guanidineHCl pH 5.0 and for the purpose of renaturation adjusted to 3 molarguanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione (reduced)and 2 mmolar glutathione (oxidized). After incubation in this solutionfor 12 hours the protein was dialyzed to 10 mmolar sodium phosphate.

[0136] The entire coding sequence including the putative signal sequenceor transmembrane domain was fused in frame with a 6-His tag present inthe expression vector pQE9 (Qiagen). E. coli harboring the expressionplasmid were induced with 1 mM IPTG during the logarithmic growth phase.Following a 3-hour induction, the cell pellet was lysed with 6MGuanidine hydrochloride and cytostatin I was purified using aNickel-chelate affinity chromatography column. The highly purifiedprotein was denatured by dialysis in PBS buffer. The gel is shown inFIG. 4: M, molecular weight markers; Lane 1 and 2, induced cell lysate;Lane 3 and 4, uninduced cell lysate; Lane 5, pass through fraction fromNickel-chelate column purification; Lane 6, 7 and 8, Fraction elutedwith 6M Guanidine hydrochloride (pH 5); 9 Fraction eluted with 6MGuanidine hydrochloride (pH 2).

EXAMPLE 2 Cloning and Expression of Cytostatin I Using the BaculovirusExpression System

[0137] The DNA sequence encoding the full length cytostatin I protein,ATCC) #97103, was amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene:

[0138] The 5′ primer has the sequence CGC GGA TCC CCC TCC CAA CCT CACTGG CTA C (SEQ ID NO:5) and contains a BamHI restriction enzyme site (inbold) followed by 22 nucleotides. The 3′ primer has the sequence CGC GGATCC CTA TCT GAC CTT CCT GAA GA (SEQ ID NO:6) and contains the cleavagesite for the restriction endonuclease BanHI and 20 nucleotides of theC-terminal coding sequence including a stop codon. The amplifiedsequences were isolated from a 1% agarose gel using a commerciallyavailable kit (“Geneclean,” BIO 101 Inc., La Jolla, Calif.). Thefragment was then digested with the endonuclease BamHI and then purifiedagain on a 1% agarose gel. This fragment is designated F2.

[0139] The vector pA2-Gp (modification of pVL941 vector, discussedbelow) is used for the expression of the cytostatin I protein using thebaculovirus expression system (for review see: Summers, M. D. and Smith,G. E. 1987, A manual of methods for baculovirus vectors and insect cellculture procedures, Texas Agricultural Experimental Station Bulletin No.1555). This expression vector contains the strong polyhedrin promoter ofthe Autographa californica nuclear polyhedrosis virus (AcMNPV) followedby the recognition sites for the restriction endonucleases BamHI. Thepolyadenylation site of the simian virus (SV)40 is used for efficientpolyadenylation. For an easy selection of recombinant virus thebeta-galactosidase gene from E.coli is inserted in the same orientationas the polyhedrin promoter followed by the polyadenylation signal of thepolyhedrin gene. The polyhedrin sequences are flanked at both sides byviral sequences for the cell-mediated homologous recombination ofco-transfected wild-type viral DNA. Many other baculovirus vectors couldbe used in place of pRG1 such as pAc373, pVL941 and pAcIMI (Luckow, V.A. and Summers, M. D., Virology, 170:31-39).

[0140] The plasmid was digested with the restriction enzymes BamHI andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The DNA was then isolated from a 1% agarose gel usingthe commercially available kit (“Geneclean” BIO 101 Inc., La Jolla,Calif.). This vector DNA is designated V2.

[0141] Fragment F2 and the dephosphorylated plasmid V2 were ligated withT4 DNA ligase. E. coli HB101 cells were then transformed and bacteriaidentified that contained the plasmid (pBac-cytostatin I) with thecytostatin I gene using the enzyme BamHI. The sequence of the clonedfragment was confirmed by DNA sequencing.

[0142] 5 μg of the plasmid pBac-cytostatin I was co-transfected with 1.0μg of a commercially available linearized baculovirus (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.) using the lipofectionmethod (Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

[0143] 1 μg of BaculoGold™ virus DNA and 5 μg of the plasmidpBac-cytostatin I were mixed in a sterile well of a microtiter platecontaining 50 μl of serum free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards 10 μl Lipofectin plus 90 μl Grace'smedium were added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture was added drop-wise to theSf9 insect cells (ATCC® CRL 1711) seeded in a 35 mm tissue culture platewith 1 ml Grace's medium without serum. The plate was rocked back andforth to mix the newly added solution. The plate was then incubated for5 hours at 27° C. After 5 hours the transfection solution was removedfrom the plate and 1 ml of Grace's insect medium supplemented with 10%fetal calf serum was added. The plate was put back into an incubator andcultivation continued at 27° C. for four days.

[0144] After four days the supernatant was collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “Blue Gal” (Life Technologies Inc.,Gaithersburg) was used which allows an easy isolation of blue stainedplaques. (A detailed description of a “plaque assay” can also be foundin the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

[0145] Four days after the serial dilution, the virus was added to thecells, blue stained plaques were picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses was thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar was removed by a brief centrifigation and the supernatantcontaining the recombinant baculovirus was used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then stored at 4° C.

[0146] Sf9 cells were grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells were infected with the recombinantbaculovirus V-cytostatin I at a multiplicity of infection (MOI) of 2.Six hours later the medium was removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham)were added. The cells were further incubated for 16 hours before theywere harvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography (FIG. 4).

EXAMPLE 3 Expression of Recombinant Cytostatin I in COS Cells

[0147] The expression of plasmid containing the cytostatin I gene isderived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 originof replication, 2) ampicillin resistance gene, 3) E. coli replicationorigin, 4) CMV promoter followed by a polylinker region, an SV40 intronand polyadenylation site. A DNA fragment encoding the entire cytostatinI precursor and a HA tag fused in frame to its 3′ end is cloned into thepolylinker region of the vector, therefore, the recombinant proteinexpression is directed under the CMV promoter. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein aspreviously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M.Connolly, and R. Lerner, 1984, Cell 37:767, (1984)). The infusion of HAtag to the target protein allows easy detection of the recombinantprotein with an antibody that recognizes the HA epitope.

[0148] The plasmid construction strategy is described as follows:

[0149] The DNA sequence encoding cytostatin I, ATCC® #97103, isconstructed by PCR on the original cytostatin I cloned using twoprimers: the 5′ primer from the 5′ end of the cytostatin I gene and a 3′sequence from the 3′ end of the cytostatin I gene. Therefore, the PCRproduct contains the acytostatin I coding sequence followed by HA tagfused in frame, a translation termination stop codon next to the HA tag,and a final restriction endonuclease site. The PCR amplified DNAfragment and the vector, pcDNAI/Amp, are digested with the appropriaterestriction enzymes and ligated. The ligation mixture is transformedinto E. coli strain SURE (available from Stratagene Cloning Systems,11099 North Torrey Pines Road, La Jolla, Calif. 92037) the transformedculture is plated on ampicillin media plates and resistant colonies areselected. Plasmid DNA is isolated from transformants and examined byrestriction analysis for the presence of the correct fragment. Forexpression of the recombinant cytostatin I, COS cells are transfectedwith the expression vector by DEAE-DEXTRAN method (J. Sambrook, E.Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, ColdSpring Laboratory Press, (1989)). The expression of the cytostatin I HAprotein is detected by radiolabelling and immunoprecipitation method (E.Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, (1988)). Cells are labelled for 8 hours with³⁵S-cysteine two days post transfection. Culture media is then collectedand cells are lysed with detergent (RJPA buffer (150 mM NaCl, 1% NP-40,0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5) (Wilson, I. et al.,Id. 37:767 (1984)). Both cell lysate and culture media are precipitatedwith an HA specific monoclonal antibody. Proteins precipitated areanalyzed on 15% SDS-PAGE gels.

EXAMPLE 4 Expression pattern of Cytostatin I in Human Tissue

[0150] Northern blot analysis is carried out to examine the levels ofexpression of cytostatin I in human tissues. Total cellular RNA samplesare isolated with RNAzol™ B system (Biotecx Laboratories Inc. 6023 SouthLoop East, Houston, Tex. 77033). About 10 μg of total RNA isolated fromeach human tissue specified is separated on 1% agarose gel and blottedonto a nylon filter (Sambrook, Fritsch, and Maniatis, Molecular Cloning,Cold Spring Harbor Press, (1989)). The labeling reaction is doneaccording to the Stratagene Prime-It kit with 50 ng DNA fragment. Thelabeled DNA is purified with a Select-G-50 column (5 Prime-3 Prime, Inc.5603 Arapahoe Road, Boulder, Colo. 80303). The filter is then hybridizedwith radioactive labeled full length cytostatin I gene at 1,000,000cpm/ml in 0.5 M NaPO₄, pH 7.4 and 7% SDS overnight at 65° C. Afterwashing twice at room temperature and twice at 60° C. with 0.5×SSC, 0.1%SDS, the filter is then exposed at −70° C. overnight with anintensifying screen. FIG. 3A issustrates the tissue distribution ofcytostatin I in various human tissues. The results are issustrated inFIGS. 3A, 3B and 3C.

EXAMPLE 5 Biological Activity of Cytostatin I

[0151] The activity of cytostatin is illustrated in FIG. 5.

[0152] Two-fold serial dilution of purified cytostatin I (MDGI homolog,HG07400-1E or HG07400-2E) starting from 100 ng/ml were made in RPMI 1640medium with 0.5% FBS. The adherent target cells were prepared fromconfluent cultures by trypsinization in PBS, and non-adherent targetcells were harvested from stationary cultures and washed once withmedium. Target cells were suspended at 1×10⁵ cells/ml in mediumcontaining 0.5 % FBS, then 0.1 ml aliquots were dispensed into 96-wellflat-bottomed microtiter plates containing 0.1 ml serially diluted testsamples. Incubation was continued for 70 hr. The activity was quantifiedusing MTS [3(4,5-dimethyl-thiazoyl-2-yl) 5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] Assay. MTSassay is performed by the addition of 20 μl of MTS and phenazinemethosulfate (PMS) solution to 96 well plates (Stock solution wasprepared as described by Promega Technical Bulletin No. 169). During a 3hr incubation, living cells convert the MTS into a the aqueous solubleformazan product. Wells with medium only (no cells) were processed inexactly the same manner as the rest of the wells and were used for blankcontrols. Wells with medium and cells were used as baseline controls.The absorbence at 490 nm was recorded using an ELISA reader and isproportional to the number of viable cells in the wells. Cell growthpromotion (positive percentage) or inhibition (negative percentage), asa percentage compared to baseline control wells (variation between threebaseline control well is less than 5%), calculated for each sampleconcentration, by the formula: OD_(experimental)/OD_(baseline control) X100−100. All determinations were made in triplicate. Mean and SD werecalculated by Microsoft Excel.

EXAMPLE 6 Expression of Cytostatin I Via Gene Therapy

[0153] Fibroblasts are obtained from a subject by skin biopsy. Theresulting tissue is placed in tissue-culture medium and separated intosmall pieces. Small chunks of the tissue are placed on a wet surface ofa tissue culture flask, approximately ten pieces are placed in eachflask. The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37° C. forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

[0154] A a selected vector such as Moloney murine leukemia virus isdigested with the appropriate restriction endonuclease and subsequentlytreated with calf intestinal phosphatase. The linear vector isfractionated on agarose gel and purified, using glass beads.

[0155] A sub-fragment of the cytostatin I cDNA is isolated and the endsof this fragment are treated with DNA polymerase in order to fill in therecessed ends and create blunt ends. Known linkers are ligated to theblunt ends with T4 DNA ligase.

[0156] Equal quantities of the Moloney murine leukemia virus linearbackbone and selected cytostatin I fragments are added together, in thepresence of T4 DNA ligase. The resulting mixture is maintained underconditions appropriate for ligation of the two fragments. The ligationmixture is used to transform bacteria HB101, which are then plated ontoagar-containing kanamycin for the purpose of confirming that the vectorhas the cytostatin I gene properly inserted.

[0157] Cytostatin I packaging cells are grown in tissue culture toconfluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10%calf serum (CS), penicillin and streptomycin. The expression vectorcontaining the cytostatin I gene is then added to the media and thepackaging cells are transduced with the vector. The packaging cells nowproduce infectious viral particles containing the cytostatin I gene (thepackaging cells are now referred to as producer cells).

[0158] Fresh media is added to the transduced producer cells, andsubsequently, media is harvested from a 10 cm plate of confluentproducer cells. The spent media, containing the infectious viralparticles, is filtered through a millipore filter to remove detachedproducer cells and this media is then used to infect fibroblast cells.Media is removed from a sub-confluent plate of fibroblasts and quicklyreplaced with the media from the producer cells . After incubation thismedia is removed and replaced with fresh media. If the titer of virus ishigh, then virtually all fibroblasts will be infected and no selectionis required. If the titer is very low, then it is necessary to use aretroviral vector that has a selectable marker, such as neo or his.

[0159] The engineered fibroblasts are then injected into the peritonealcavity of rats, either alone or after having been grown to confluence oncytodex 3 microcarrier beads. The fibroblasts now produce the cytostatinI protein product.

[0160] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, within thescope of the appended claims, the invention may be practiced otherwisethan as particularly described.

1 11 1 861 DNA human CDS (94)..(414) 1 cacgagctgg aatctctcag cctcacctgccagacaacac cccctccttc ctcaccctgt 60 ttcctgcatt ctcctgaaac cttcatccac acaatg cct ccc aac ctc act ggc 114 Met Pro Pro Asn Leu Thr Gly 1 5 tac taccgc ttt gtt tcg cag aag aac atg gag gac tac ctg caa gcc 162 Tyr Tyr ArgPhe Val Ser Gln Lys Asn Met Glu Asp Tyr Leu Gln Ala 10 15 20 cta aac atcagc ttg gct gtg cgg aag atc gcg ctg ctg ctg aag ccg 210 Leu Asn Ile SerLeu Ala Val Arg Lys Ile Ala Leu Leu Leu Lys Pro 25 30 35 gac aag gag atcgaa cac cag ggc aac cac atg acg gtg agg acg ctc 258 Asp Lys Glu Ile GluHis Gln Gly Asn His Met Thr Val Arg Thr Leu 40 45 50 55 agc acc ttc cgaaac tac act ttg cag ttt gat gtg gga gtg cag aaa 306 Ser Thr Phe Arg AsnTyr Thr Leu Gln Phe Asp Val Gly Val Gln Lys 60 65 70 ggg gag gtc ccc aaccgg ggc tgg aga cac tgg ctg gag gga gag ttg 354 Gly Glu Val Pro Asn ArgGly Trp Arg His Trp Leu Glu Gly Glu Leu 75 80 85 ctg tat ctg gaa ctg actgca agg gat gca gtg tgc gag cag gtc ttc 402 Leu Tyr Leu Glu Leu Thr AlaArg Asp Ala Val Cys Glu Gln Val Phe 90 95 100 agg aag gtc aga tagccggagaggagccaaga tccctccaga cagcaccagc 454 Arg Lys Val Arg 105 tcacagacgctcttgttgtg cccccttcaa gcccagattg tgccaggtca gctgtccctt 514 cctctggccacctttcctcc ctctgggtcc ctcctcaccc ctccccgtgt taatctgtaa 574 cttggagcccccaggacaaa gtcctttctc acactccact gcccaatagt gacctcactt 634 ccaggtcaaggtctggcgtc ccaaatgaaa gaagcaggca aagggaagga gcccctgagg 694 acaaccaatctccgctctct cctgtccatt tgacctcttc ttttccttct aagaaagaac 754 taagctttgggcatttggcg attagtgaaa attctatcct gatggacttc tggaaaactg 814 tgactggggttcaacagttt aaacaggggc tactggggga aaaaaaa 861 2 107 PRT human 2 Met ProPro Asn Leu Thr Gly Tyr Tyr Arg Phe Val Ser Gln Lys Asn 1 5 10 15 MetGlu Asp Tyr Leu Gln Ala Leu Asn Ile Ser Leu Ala Val Arg Lys 20 25 30 IleAla Leu Leu Leu Lys Pro Asp Lys Glu Ile Glu His Gln Gly Asn 35 40 45 HisMet Thr Val Arg Thr Leu Ser Thr Phe Arg Asn Tyr Thr Leu Gln 50 55 60 PheAsp Val Gly Val Gln Lys Gly Glu Val Pro Asn Arg Gly Trp Arg 65 70 75 80His Trp Leu Glu Gly Glu Leu Leu Tyr Leu Glu Leu Thr Ala Arg Asp 85 90 95Ala Val Cys Glu Gln Val Phe Arg Lys Val Arg 100 105 3 28 DNA human 3cgcggatcca tgcctcccaa cctcactg 28 4 30 DNA human 4 gcgtctagac tatctgaccttcctgaagac 30 5 31 DNA human 5 cgcggatccc cctcccaacc tcactggcta c 31 629 DNA human 6 cgcggatccc tatctgacct tcctgaaga 29 7 133 PRT human 7 MetAla Asp Ala Phe Val Gly Thr Trp Lys Leu Val Asp Ser Lys Asn 1 5 10 15Phe Asp Asp Tyr Met Lys Ser Leu Gly Val Gly Phe Ala Thr Arg Gln 20 25 30Val Ala Ser Met Thr Lys Pro Thr Thr Ile Ile Glu Lys Asn Gly Asp 35 40 45Thr Ile Thr Ile Lys Thr Gln Ser Thr Phe Lys Asn Thr Glu Ile Asn 50 55 60Phe Gln Leu Gly Ile Glu Phe Asp Glu Val Thr Ala Asp Asp Arg Lys 65 70 7580 Val Lys Ser Leu Val Thr Leu Asp Gly Gly Lys Leu Ile His Val Gln 85 9095 Lys Trp Asn Gly Gln Glu Thr Thr Leu Thr Arg Glu Leu Val Asp Gly 100105 110 Lys Leu Ile Leu Thr Leu Thr His Gly Ser Val Val Ser Thr Arg Thr115 120 125 Tyr Glu Lys Glu Ala 130 8 135 PRT human 8 Met Pro Val AspPhe Thr Gly Tyr Trp Lys Met Leu Val Asn Glu Asn 1 5 10 15 Phe Glu GluTyr Leu Arg Ala Leu Asp Val Asn Val Ala Leu Arg Lys 20 25 30 Ile Ala AsnLeu Leu Lys Pro Asp Lys Glu Ile Val Gln Asp Gly Asp 35 40 45 His Met IleIle Arg Thr Leu Ser Thr Phe Arg Asn Tyr Ile Met Asp 50 55 60 Phe Gln ValGly Lys Glu Phe Glu Glu Asp Leu Thr Gly Ile Asp Asp 65 70 75 80 Arg LysCys Met Thr Thr Val Ser Trp Asp Gly Asp Lys Leu Gln Cys 85 90 95 Val GlnLys Gly Glu Lys Glu Gly Arg Gly Trp Thr Gln Trp Ile Glu 100 105 110 GlyAsp Glu Leu His Leu Glu Met Arg Val Glu Gly Val Val Cys Lys 115 120 125Gln Val Phe Lys Lys Val Gln 130 135 9 134 PRT human 9 Met Thr Arg AspGln Asn Gly Thr Trp Glu Met Glu Ser Asn Glu Asn 1 5 10 15 Phe Glu GlyTyr Met Lys Ala Leu Asp Ile Asp Phe Ala Thr Pro Lys 20 25 30 Ile Ala ValArg Leu Thr Gln Thr Lys Val Ile Asp Gln Asp Gly Asp 35 40 45 Asn Phe LysThr Lys Thr Thr Ser Thr Phe Arg Asn Tyr Asp Val Asp 50 55 60 Phe Thr ValGly Val Glu Phe Asp Glu Tyr Thr Lys Ser Leu Asp Asn 65 70 75 80 Arg HisVal Lys Ala Leu Val Thr Trp Glu Gly Asp Val Leu Val Cys 85 90 95 Val GlnLys Gly Glu Lys Glu Asn Arg Gly Trp Lys Gln Trp Ile Glu 100 105 110 GlyAsp Lys Leu Tyr Leu Glu Leu Thr Cys Gly Asp Gln Val Cys Arg 115 120 125Gln Val Phe Lys Lys Lys 130 10 133 PRT human 10 Met Val Asp Ala Phe LeuGly Thr Trp Lys Leu Val Asp Ser Lys Asn 1 5 10 15 Phe Asp Asp Tyr MetLys Ser Leu Gly Val Gly Phe Ala Thr Arg Gln 20 25 30 Val Ala Ser Met ThrLys Pro Thr Thr Ile Ile Glu Lys Asn Gly Asp 35 40 45 Ile Leu Thr Leu LysThr His Ser Thr Phe Lys Asn Thr Glu Ile Ser 50 55 60 Phe Lys Leu Gly ValGlu Phe Asp Glu Thr Thr Ala Asp Asp Arg Lys 65 70 75 80 Val Lys Ser IleVal Thr Leu Asp Gly Gly Lys Leu Val His Leu Gln 85 90 95 Lys Trp Asp GlyGln Glu Thr Thr Leu Val Arg Glu Leu Ile Asp Gly 100 105 110 Lys Leu IleLeu Thr Leu Thr His Gly Thr Ala Val Cys Thr Arg Thr 115 120 125 Tyr GluLys Glu Ala 130 11 132 PRT human 11 Met Ser Asn Lys Phe Leu Gly Thr TrpLys Leu Val Ser Ser Glu Asn 1 5 10 15 Phe Asp Asp Tyr Met Lys Ala LeuGly Val Gly Leu Ala Thr Arg Lys 20 25 30 Leu Gly Asn Leu Ala Lys Pro ThrVal Ile Ile Ser Lys Lys Gly Asp 35 40 45 Ile Ile Thr Ile Arg Thr Glu SerThr Phe Lys Asn Thr Glu Ile Ser 50 55 60 Phe Lys Leu Gly Gln Glu Phe GluGlu Thr Thr Ala Asp Asn Arg Lys 65 70 75 80 Thr Lys Ser Ile Val Thr LeuGln Arg Gly Ser Leu Asn Gln Val Gln 85 90 95 Arg Trp Asp Gly Lys Glu ThrThr Ile Lys Arg Lys Leu Val Asn Gly 100 105 110 Lys Met Val Ala Glu CysLys Met Lys Gly Val Val Cys Thr Arg Ile 115 120 125 Tyr Glu Lys Val 130

What is claimed is:
 1. An isolated antibody or portion thereof thatspecifically binds to a protein whose sequence consists of amino acidresidues +1 to +107 of SEQ ID NO:2.
 2. The antibody or portion thereofof claim 1 wherein said protein specifically bound by said antibody orportion thereof is glycosylated.
 3. The antibody or portion thereof ofclaim 1 which is a monoclonal antibody.
 4. The antibody or portionthereof of claim 1 which is a polyclonal antibody.
 5. The antibody orportion thereof of claim 1 which is a chimeric antibody.
 6. The antibodyor portion thereof of claim 1 which is a single chain antibody.
 7. Theantibody or portion thereof of claim 1 which is a Fab fragment.
 8. Theantibody or portion thereof of claim 1 which is labeled.
 9. The antibodyof claim 8 wherein the label is selected from the group consisting of:(a) an enzyme label; (b) a radioisotope; and (c) a fluorescent label.10. A composition comprising the antibody or portion thereof of claim 1and a carrier.
 11. The composition of claim 10, wherein the antibody orportion thereof is a monoclonal antibody.
 12. The composition of claim10, wherein the antibody or portion thereof is a polyclonal antibody.13. The composition of claim 10, wherein the antibody or portion thereofis a chimeric antibody.
 14. The composition of claim 10, wherein theantibody or portion thereof is a single chain antibody.
 15. Thecomposition of claim 10, wherein the antibody or portion thereof is aFab fragment.
 16. The composition of claim 10, wherein the antibody orportion thereof is labeled.
 17. The composition of claim 16 wherein thelabel is selected from the group consisting of: (a) an enzyme label; (b)a radioisotope; and (c) a fluorescent label.
 18. An isolated cell thatproduces the antibody of claim
 1. 19. A hybridoma that produces theantibody of claim
 1. 20. A hybridoma that produces the antibody of claim3.
 21. A method of detecting Cytostatin I protein in a biological samplecomprising: (a) contacting the biological sample with the antibody orportion thereof of claim 1; and (b) detecting the Cytostatin I proteinin the biological sample.
 22. The method of claim 21 wherein theantibody is a monoclonal antibody.
 23. The method of claim 21 whereinthe antibody is a polyclonal antibody.
 24. The method of claim 21wherein the antibody is a chimeric antibody.
 25. The method of claim 21wherein the antibody is a single chain antibody.
 26. The method of claim21 wherein the antibody is a Fab fragment.
 27. The method of claim 21wherein the antibody is a labeled antibody.
 28. The method of claim 27wherein the label is selected from the group consisting of: (a) anenzyme label; (b) a radioisotope; and (c) a fluorescent label.
 29. Anisolated antibody or portion thereof produced by immunizing an animalwith a protein whose sequence comprises amino acid residues +1 to +107of SEQ ID NO:2; wherein said antibody or portion thereof specificallybinds to the amino acid sequence of SEQ ID NO:2.
 30. An isolatedantibody or portion thereof that specifically binds to a proteinselected from the group consisting of: (a) a protein whose sequenceconsists of amino acid residues +39 to +107 of SEQ ID NO:2; (b) aprotein whose sequence consists of amino acid residues +22 to +107 ofSEQ ID NO:2; (c) a protein whose sequence consists of 30 contiguousamino acid residues of SEQ ID NO:2; and (d) a protein whose sequenceconsists of 50 contiguous amino acid residues of SEQ ID NO:2.
 31. Theisolated antibody or portion thereof of claim 30, that specificallybinds protein (a).
 32. The isolated antibody or portion thereof of claim30, that specifically binds protein (b).
 33. The isolated antibody orportion thereof of claim 30, that specifically binds protein (c). 34.The isolated antibody or portion thereof of claim 30, that specificallybinds protein (d).
 35. The isolated antibody or portion thereof of claim30, wherein said protein specifically bound by said isolated antibody orportion thereof is glycosylated.
 36. The isolated antibody or portionthereof of claim 30 which is a monoclonal antibody.
 37. The isolatedantibody or portion thereof of claim 30 which is a polyclonal antibody.38. The isolated antibody or portion thereof of claim 30, which is achimeric antibody.
 39. The isolated antibody or portion thereof of claim30 which is a single chain antibody.
 40. The isolated antibody orportion thereof of claim 30 which is a Fab fragment.
 41. The antibody orportion thereof of claim 30 which is labeled.
 42. The antibody of claim41 wherein the label is selected from the group consisting of: (a) anenzyme label; (b) a radioisotope; and (c) a fluorescent label.
 43. Acomposition comprising the isolated antibody or portion thereof of claim30 and a carrier.
 44. The composition of claim 43, wherein the isolatedantibody or portion thereof is a monoclonal antibody.
 45. Thecomposition of claim 43, wherein the isolated antibody or portionthereof is a polyclonal antibody.
 46. The composition of claim 43,wherein the isolated antibody or portion thereof is a chimeric antibody.47. The composition of claim 43, wherein the isolated antibody orportion thereof is a single chain antibody.
 48. The composition of claim43, wherein the isolated antibody or portion thereof is a Fab fragment.49. The composition of claim 43, wherein the antibody or portion thereofis labeled.
 50. The composition of claim 49 wherein the label isselected from the group consisting of: (a) an enzyme label; (b) aradioisotope; and (c) a fluorescent label.
 51. An isolated cell thatproduces the antibody or portion thereof of claim
 30. 52. A hybridomathat produces the monoclonal antibody of claim
 30. 53. A hybridoma thatproduces the monoclonal antibody of claim
 36. 54. A method of assayingCytostatin I protein in a biological sample comprising: (a) contactingthe biological sample with the isolated antibody or portion thereof ofclaim 30; and (b) detecting Cytostatin I protein in the biologicalsample.
 55. The method of claim 54 wherein the isolated antibody orportion thereof is a monoclonal antibody.
 56. The method of claim 54wherein the isolated antibody or portion thereof is a polyclonalantibody.
 57. The method of claim 54 wherein the isolated antibody orportion thereof is a chimeric antibody.
 58. The method of claim 54wherein the isolated antibody or portion thereof is a single chainantibody.
 59. The method of claim 54 wherein the isolated antibody orportion thereof is a Fab fragment.
 60. The method of claim 54 whereinthe isolated antibody or portion thereof is a labeled antibody.
 61. Themethod of claim 60 wherein the label is selected from the groupconsisting of: (a) an enzyme label; (b) a radioisotope; and (c) afluorescent label.
 62. An antibody or portion thereof produced byimmunizing an animal with a protein selected from the group consistingof: (a) a protein whose sequence comprises amino acid residues +39 to+107 of SEQ ID NO:2; (b) a protein whose sequence comprises amino acidresidues +22 to +107 of SEQ ID NO:2; (c) a protein whose sequencecomprises 30 contiguous amino acid residues of SEQ ID NO:2; and (d) aprotein whose sequence comprises 50 contiguous amino acid residues ofSEQ ID NO:2; wherein said antibody or portion thereof specifically bindsto the amino acid sequence of SEQ ID NO:2.
 63. The antibody or portionthereof of claim 62 produced by immunizing an animal with protein (a).64. The antibody or portion thereof of claim 62 produced by immunizingan animal with protein (b).
 65. The antibody or portion thereof of claim62 produced by immunizing an animal with protein (c).
 66. The antibodyor portion thereof of claim 62 produced by immunizing an animal withprotein (d).
 67. An isolated antibody or portion thereof thatspecifically binds to a protein whose sequence consists of the aminoacid sequence of the polypeptide encoded by the cDNA contained in ATCC®)Deposit No.
 97103. 68. The antibody or portion thereof of claim 67wherein said protein specifically bound by said antibody or portionthereof is glycosylated.
 69. The antibody or portion thereof of claim 67which is a monoclonal antibody.
 70. The antibody or portion thereof ofclaim 67 which is a polyclonal antibody.
 71. The antibody or portionthereof of claim 67 which is a chimeric antibody.
 72. The antibody orportion thereof of claim 67 which is a single chain antibody.
 73. Theantibody or portion thereof of claim 67 which is a Fab fragment.
 74. Theantibody or portion thereof of claim 67 which is labeled.
 75. Theantibody of claim 74 wherein t he label is selected from the groupconsisting of: (a) an enzyme label; (b) a radioisotope; and (c) afluorescent label.
 76. A composition comprising the antibody or portionthereof of claim 67 and a carrier.
 77. The composition of claim 76,wherein the antibody or portion thereof is a monoclonal antibody. 78.The composition of claim 76, wherein the antibody or portion thereof isa polyclonal antibody.
 79. The composition of claim 76, wherein theantibody or portion thereof is a chimeric antibody.
 80. The compositionof claim 76, wherein the antibody or portion thereof is a single chainantibody.
 81. The composition of claim 76, wherein the antibody orportion thereof is a Fab fragment.
 82. The composition of claim 76,wherein the antibody or portion thereof is labeled.
 83. The compositionof claim 82 wherein the label is selected from the group consisting of:(a) an enzyme label; (b) a radioisotope; and (c) a fluorescent label.84. An isolated cell that produces the antibody of claim
 67. 85. Ahybridoma that produces the antibody of claim
 67. 86. A hybridoma thatproduces the antibody of claim
 69. 87. A method of detecting CytostatinI protein in a biological sample comprising: (a) contacting thebiological sample with the antibody or portion thereof of claim 67; and(b) detecting the Cytostatin I protein in the biological sample.
 88. Themethod of claim 87 wherein the antibody is a monoclonal antibody. 89.The method of claim 87 wherein the antibody is a polyclonal antibody.90. The method of claim 87 wherein the antibody is a chimeric antibody.91. The method of claim 87 wherein the antibody is a single chainantibody.
 92. The method of claim 87 wherein the antibody is a Fabfragment.
 93. The method of claim 87 wherein the antibody is a labeledantibody.
 94. The method of claim 93 wherein the label is selected fromthe group consisting of: (a) an enzyme label; (b) a radioisotope; and(c) a fluorescent label.
 95. An antibody or portion thereof produced byimmunizing an animal with a protein whose sequence comprises the aminoacid sequence of the polypeptide encoded by the cDNA contained in ATCC®Deposit No. 97103; wherein said antibody or portion thereof specificallybinds to the amino acid sequence of the polypeptide encoded by the cDNAcontained in ATCC® Deposit No.
 97103. 96. An isolated antibody orportion thereof that specifically binds to a protein selected from thegroup consisting of: (a) a protein whose sequence consists of the aminoacid sequence of the mature form of the polypeptide encoded by the cDNAcontained in ATCC® Deposit No. 97103; (b) a protein whose sequenceconsists of at least 30 contiguous amino acid residues of a polypeptideencoded by the cDNA contained in ATCC®) Deposit No. 97103; and (c) aprotein whose sequence consists of at least 50 contiguous amino acidresidues of a polypeptide encoded by the cDNA contained in ATCC® DepositNo.
 97103. 97. The isolated antibody or portion thereof of claim 96 thatspecifically binds protein (a).
 98. The isolated antibody or portionthereof of claim 96 that specifically binds protein (b).
 99. Theisolated antibody or portion thereof of claim 96 that specifically bindsprotein (c).
 100. The isolated antibody or portion thereof of claim 96,wherein said protein specifically bound by said antibody or portionthereof is glycosylated.
 101. The isolated antibody or portion thereofof claim 96, which is a monoclonal antibody.
 102. The isolated antibodyor portion thereof of claim 96, which is a polyclonal antibody.
 103. Theisolated antibody or portion thereof of claim 96, which is a chimericantibody.
 104. The isolated antibody or portion thereof of claim 96which is a single chain antibody.
 105. The isolated antibody or portionthereof of claim 96 which is a Fab fragment.
 106. The isolated antibodyor portion thereof of claim 96 which is labeled.
 107. The isolatedantibody or portion thereof of claim 106 wherein the label is selectedfrom the group consisting of: (a) an enzyme label; (b) a radioisotope;and (c) a fluorescent label.
 108. A composition comprising the isolatedantibody or portion thereof of claim 96 and a carrier.
 109. Thecomposition of claim 108, wherein the antibody or portion thereof is amonoclonal antibody.
 110. The composition of claim 108, wherein theantibody or portion thereof is a polyclonal antibody.
 111. Thecomposition of claim 108, wherein the antibody or portion thereof is achimeric antibody.
 112. The composition of claim 108, wherein theantibody or portion thereof is a single chain antibody.
 113. Thecomposition of claim 108, wherein the antibody or portion thereof is aFab fragment.
 114. An isolated cell that produces the antibody orportion thereof of claim
 96. 115. A hybridoma that produces themonoclonal antibody of claim
 96. 116. A hybridoma that produces themonoclonal antibody of claim
 101. 117. A method of assaying Cytostatin Iprotein in a biological sample comprising: (a) contacting the biologicalsample from a test subject with the isolated antibody or portion thereofof claim 96; and (b) detecting Cytostatin I protein in the biologicalsample.
 118. The method of claim 117, wherein the antibody or portionthereof is a monoclonal antibody.
 119. The method of claim 117, whereinthe antibody or portion thereof is a polyclonal antibody.
 120. Themethod of claim 117, wherein the antibody or portion thereof is achimeric antibody.
 121. The method of claim 117, wherein the antibody orportion thereof is a single chain antibody.
 122. The method of claim117, wherein the antibody or portion thereof is a Fab fragment.
 123. Anantibody or portion thereof produced by immunizing an animal with aprotein selected from the group consisting of: (a) a protein whosesequence comprises the amino acid sequence of the mature form of thepolypeptide encoded by the cDNA contained in ATCC® Deposit Number 97103;(b) a protein whose sequence comprises at least 30 contiguous amino acidresidues of a polypeptide encoded by the cDNA contained in ATCC® DepositNo. 97103; and (c) a protein whose sequence comprises at least 50contiguous amino acid residues of a polypeptide encoded by the cDNAcontained in ATCC® Deposit No. 97103; wherein said antibody or portionthereof specifically binds to the polypeptide encoded by the cDNAcontained in ATCC® Deposit No.
 97103. 124. (New) The antibody or portionthereof of claim 123 produced by immunizing an animal with protein (a).125. (New) The antibody or portion thereof of claim 123 produced byimmunizing an animal with protein (b).
 126. (New) The antibody orportion thereof of claim 123 produced by immunizing an animal withprotein (c).
 127. A method of treating a patient having need ofCytostatin I protein.
 128. A method of treating a patient having need ofa reduced level of Cytostatin I protein, comprising administering tosaid patient the antibody or portion thereof of claim
 1. 129. A methodof treating a patient having need of a reduced level of Cytostatin Iprotein, comprising administering to said patient the antibody orportion thereof of claim
 30. 130. A method of treating a patient havingneed of a reduced level of Cytostatin I protein, comprisingadministering to said patient the antibody or portion thereof of claim67.
 131. A method of treating a patient having need of a reduced levelof Cytostatin I protein, comprising administering to said patient theantibody or portion thereof of claim 96.